Expression of nestin after renal transplantation in the rat

Chronic allograft injury (CAI) limits the long‐term success of renal transplantation. Nestin is a marker of progenitor cells, which probably contribute to its pathogenesis. We hypothesize that nestin is induced by ischemia/reperfusion injury and acute rejection, main risk factors for CAI. Syngeneic renal transplantation was performed in Lewis rats and allogeneic transplantation in the Fischer 344 to Lewis strain combination, which results in reversible acute rejection and in CAI in the long‐run. The Dark Agouti to Lewis rat strain combination was used to study fatal acute rejection. In untreated kidneys, nestin immunoreactivity was detected in glomeruli and in very few interstitial or microvascular cells. Syngeneic transplantation induced nestin expression within 4 days, which decreased until day 9 and returned to control levels on day 42. Nestin expression was strong during acute rejection and still detected during the pathogenesis of CAI on day 42. Nestin‐positive cells were identified as endothelial cells and interstitial fibroblast‐like cells co‐expressing alpha‐smooth muscle actin. A sub‐population of them expressed proliferating cell nuclear antigen. In conclusion, nestin is induced in renal grafts by ischemia/reperfusion injury and acute rejection. It is expressed by proliferating myofibroblasts and endothelial cells and probably contributes to the pathogenesis of CAI.     

Differential immunoexpressions of cytoskeletons in renal epithelial and interstitial cells in rat and canine fibrotic kidneys, and in kidney-related cell lines under fibrogenic stimuli

Myofibroblasts play an important role in chronic renal interstitial fibrosis. However, the origin and developmental mechanisms remain to be elucidated. The myofibroblasts may express various cytoskeletons during the development. Immunoexpressions of vimentin, desmin and alpha-smooth muscle actin (alpha-SMA) were analyzed using experimentally (cisplatin and unilateral ureteral obstruction) induced rat and spontaneous canine fibrotic kidneys or kidney-related cell lines incubated with transforming growth factor-beta1 (TGF-beta1), platelet-derived growth factor-BB (PDGF-BB) or their combination at various concentrations. In rat fibrotic kidneys, both renal epithelia and interstitial cells showed positive reactions to alpha-SMA and vimentin, supporting epithelial-mesenchymal transition (EMT) theory; however, renal epithelia did not react to desmin, though interstitial cells were reactive. Renal epithelia in canine fibrotic kidneys did not show a positive reaction to alpha-SMA, whereas interstitial cells reacted strongly to alpha-SMA; conversely, renal epithelia reacted strongly to desmin, but interstitial cells did not; vimentin expression was infrequently seen in renal epithelia and interstitial cells of canine kidneys. Exposure of TGF-beta1 to porcine renal epithelial cells (LLC-PK1), rat renal interstitial cells (NRK-49F), and rat immature mesenchymal cells (MT-9) dose-dependently increased selectively alpha-SMA-positive cell numbers. Moreover, PDGF-BB exhibited an additive effect on TGF-beta1-induced alpha-SMA expression in these cell lines when simultaneously added. alpha-SMA was the most plastic cytoskeleton under fibrogenic stimuli. This study shows that there are interspecies differences in cytoskeletal immunoexpressions of renal epithelia or interstitial cells between rat and canine fibrotic kidneys, and that the derivation of renal myofibroblasts may be heterogeneous, such as renal epithelia, interstitial cells or immature mesenchymal cells.     

Diabetes induces changes in glomerular development and laminin-beta 2 (s-laminin) expression.

Offspring of diabetic mothers have developmental renal abnormalities; thus, we investigated the effects of the diabetic milieu on kidney development. Four groups of host rats, including insulin-deficient and insulin-treated streptozotocin-induced diabetic rats, normal controls, and insulin-treated nondiabetic rats, were prepared. After 38 days, rats received ocular implants of E14 fetal rat kidneys. Nine days later the fetal kidney grafts were harvested for analysis of glomerular development and expression of fibronectin, laminin, laminin-beta 2, and alpha-smooth muscle actin and m170, two additional markers of mesangial maturation. The rate of glomerular maturation was delayed in grafts placed in hyperglycemic, insulin-deficient diabetic rats. These glomeruli contained few mesangial cells or matrix, and laminin-beta 2 expression was reduced as compared with controls. Mesangial expression of alpha-smooth muscle actin and m170 was not detected. In contrast, grafts placed in insulin-treated diabetic animals had increased numbers of mesangial cells and expanded mesangial matrix. The content of laminin-beta 2 and expression of m170 and alpha-smooth muscle actin were also increased in these grafts. These data show that hyperglycemia and insulin status influence laminin isoform expression and play important roles in mesangial development.     

The role of cell plasticity in progression and reversal of renal fibrosis

The need for novel insights into the mechanisms of progression of renal disease has become urgent during the last several years because of the increasing incidence of chronic renal disease worldwide. Independent of the underlying disease, the subsequent progression of renal fibrosis is characterized mainly by both an exaggerated synthesis and abnormal accumulation of extracellular matrix proteins produced by mesenchymal cells within the kidney. These cells are mainly myofibroblasts deriving from a variety of renal cells such as vascular smooth muscle, mesangial, resident stem, tubular epithelial, vascular endothelial cells or pericytes. The appearance of myofibroblasts is a reversible process, as suggested by studies in experimental models showing regression of renal fibrosis during therapy with antagonists and/or blockers of the renin-angiotensin system. An additional factor that can also affect the mechanisms of progression/regression of fibrosis is the plasticity of podocytes controlling glomerular filtration.     

Myofibroblasts in experimental hydronephrosis.

Interstitial fibrosis is a common outcome of longterm ureteral obstruction. One pathological arm of the fibrotic reaction in diverse tissue loci and experimental models is the retraction of granulation tissue. The role of the myofibroblast in granulation tissue contraction and fibrocontractive diseases has been well established, but the mechanisms leading to differentiation of fibroblastic cells into myofibroblasts during the evolution of inflammation are not yet fully clarified. Investigators using other model systems have shown that macrophage-derived transforming growth factor-beta 1 (TGF-beta 1) may be pivotal in the process of myofibroblast modulation. Our laboratory has shown that the unilateral ureteral obstruction in the rat is characterized by a 20-fold increment in infiltrating renal cortical interstitial macrophages, an increase in cortical TGF-beta 1 gene expression, which parallels the infiltrating macrophage burden, and immunolocalization of this peptide growth factor in close proximity to resident interstitial fibroblasts. Because of this model's features, it was our aim to assess whether a myofibroblastic modulation was operant in the renal cortex of obstructed rat kidneys versus the control contralateral unobstructed kidney specimens. Immunolabeling for alpha-smooth muscle actin and the intermediate filament protein, desmin, was detected and steadily intensified from 24 to 96 hours after unilateral ureteral obstruction in obstructed kidneys only. In temporal concert with the detection of alpha-smooth muscle actin protein, the mRNA expression for this cytoskeletal component exhibited 3.7-, 15.7-, and 4.1-fold increments in the renal cortex of obstructed kidneys versus the contralateral unobstructed kidney specimens at 24, 48, and 96 hours after unilateral ureteral obstruction, respectively. Whole body X-irradiation, administered to rats 11 days before proximal left ureteral ligation, significantly lowered cortical interstitial macrophage number, cortical TGF-beta and alpha-smooth muscle actin mRNA levels as well as the intensity of immunolabeling for alpha-smooth muscle actin from 12 to 96 hours after unilateral ureteral obstruction. These data support a postulate that renal cortical TGF-beta 1, derived from the infiltrating macrophage, in part, contributes to the subsequent interstitial fibrosis response to renal injury by fostering the modulation of fibroblasts to myofibroblasts within the renal cortex after ureteral obstruction.     

Pentraxin 3 Inhibits Acute Renal Injury-Induced Interstitial Fibrosis Through Suppression of IL-6/Stat3 Pathway

Acute kidney injury-induced organ fibrosis is recognized as a major risk factor for the development of chronic kidney disease, which remains one of the leading causes of death in the developed world. However, knowledge on molecules that may suppress the fibrogenic response after injury is lacking. The long pentraxin 3 (PTX3), a novel acute renal injury marker, has been reported to be involved in chronic renal injury, but the mechanism is still unknown. In this experiment, the mice subjected to acute kidney injury showed a slow recovery of kidney function compared with PTX3-treated animals. Collagen expression was absent in sham-operated kidneys; however, their expression was significantly increased after reperfusion. And, these changes were reduced in PTX3-treated mouse kidney. Fibrosis was associated with increased expression of IL-6 and extensive activation of Stat3. Administration of IL-6 increased collagen I expression and Stat3 activation in vitro in renal epithelial cells subjected to hypoxia-reoxygenation, which was suppressed by PTX3. Furthermore, we found that the decreased serum creatinine level and the reduced expression of collagen and smooth muscle actin induced by PTX3 were abolished by additional administration of IL-6. The associated p-Stat3 expression which was reduced by PTX3 administration was also inverted by additional IL-6 treatment. Our data suggest that PTX3 inhibits acute renal injury-induced interstitial fibrosis through suppression of IL-6/Stat3 pathway.     

Thy-1 expression,a possible marker of early myofibroblast development,in renal tubulointerstitial fibrosis induced in rats by cisplatin

Interstitial fibrosis is regarded as the common final pathway in chronic renal failure. Myofibroblasts play an important role in the renal fibrosis through producing extracellular matrices. In addition to expressions of cytoskeletons such as vimentin, desmin and α-smooth muscle actin (α-SMA), Thy-1 expression was investigated in cisplatin-induced rat renal interstitial fibrosis, to clarify the characteristics of myofibroblasts. Immunohistochemically, myofibroblasts in the renal fibrotic lesions reacted to vimentin, desmin and α-SMA in varying degrees, and the expression degrees were increased with advancing fibrosis. Vimentin expression was the greatest and the increased expression retained even in scar at end stages, whereas desmin and α-SMA expressions were almost completely decreased in scar. In double immunofluorescence, there were myofibroblasts reacting to both vimentin/desmin, desmin/α-SMA or α-SMA/vimentin, indicating that renal myofibroblasts can simultaneously express different cytoskeletons. Thy-1 expression in renal myofibroblasts was increased according to progressing fibrosis; however, the increased expression was decreased in scar, similar to desmin and α-SMA expressions. Some myofibroblasts expressing Thy-1 reacted simultaneously to vimentin or desmin, but there were no cells reacting to both Thy-1 and α-SMA. Because well-differentiated myofibroblasts are characterized mainly by α-SMA expression and the pericytes (immature stromal stem cells) showed a positive reaction to Thy-1, renal myofibroblasts might be originated from immature mesenchymal cells through loosing Thy-1 expression. This study for the first time shows that renal myofibroblasts can variously exhibit such mesenchymal markers as vimentin, desmin, α-SMA and Thy-1; particularly, Thy-1 immunohistochemistry would be used to detect myofibroblasts at early stages in analyzing chemically induced renal lesions.     

The contribution of bone marrow-derived cells to the development of renal interstitial fibrosis

Recent evidence suggests that bone marrow (BM)-derived cells may integrate into the kidney, giving rise to functional renal cell types, including endothelial and epithelial cells and myofibroblasts. BM-derived cells can contribute to repair of the renal peritubular capillary (PTC) network following acute ischemic injury. However, the cell fate and regulation of BM-derived cells during the progression of chronic renal disease remains unclear. Using chimeric mice transplanted with enhanced green fluorescent protein (EGFP)-expressing BM, we demonstrate that the number of BM-derived myofibroblasts coincided with the development of fibrosis in a mouse adriamycin (ADR)-induced nephrosis model of chronic, progressive renal fibrosis. Four weeks after ADR injection, increased numbers of BM-derived myofibroblasts were observed in the interstitium of ADR-injected mice. Six weeks after ADR injection, more than 30% of renal alpha-smooth muscle actin (+) (alpha-SMA+) interstitial myofibroblasts were derived from the BM. In addition, BM-derived cells were observed to express the endothelial cell marker CD31 and the myofibroblast marker alpha-SMA. Blockade of p38 mitogen-activated protein kinase (MAPK) and transforming growth factor (TGF)-beta1/Smad2 signaling was found to protect BM-derived PTC endothelial cells and inhibit the number of BM-derived von Willebrand factor (vWF)(+)/EGFP(+)/alpha-SMA(+) cells, EGFP(+)/alpha-SMA(+) cells, and total alpha-SMA(+) cells in ADR-injected mice. Inhibition of the p38 MAPK and TGF-beta1/Smad signaling pathways enhanced PTC repair by decreasing endothelial-myofibroblast transformation, leading to structural and functional renal recovery and the attenuation of renal interstitial fibrosis. Investigation of the signaling pathways that regulate the differentiation and survival of BM-derived cells in a progressive disease setting is vital for the successful development of cell-based therapies for renal repair.     

Conditional ablation of macrophages halts progression of crescentic glomerulonephritis

The presence of macrophages in inflamed glomeruli of rat kidney correlates with proliferation and apoptosis of resident glomerular mesangial cells. We assessed the contribution of inflammatory macrophages to progressive renal injury in murine crescentic glomerulonephritis (GN). Using a novel transgenic mouse (CD11b-DTR) in which tissue macrophages can be specifically and selectively ablated by minute injections of diphtheria toxin, we depleted renal inflammatory macrophages through days 15 and 20 of progressive crescentic GN. Macrophage depletion reduced the number of glomerular crescents, improved renal function, and reduced proteinuria. Morphometric analysis of renal tubules and interstitium revealed a marked attenuation of tubular injury that was associated with reduced proliferation and apoptosis of tubular cells. The population of interstitial myofibroblasts decreased after macrophage depletion and interstitial fibrosis also decreased. In the presence of macrophages, interstitial myofibroblasts exhibited increased levels of both proliferation and apoptosis, suggesting that macrophages act to support a population of renal myofibroblasts in a high turnover state and in matrix deposition. Finally, deletion of macrophages reduced CD4 T cells in the diseased kidney. This study demonstrates that macrophages are key effectors of disease progression in crescentic GN, acting to regulate parenchymal cell populations by modulating both cell proliferation and apoptosis.     

单侧输尿管梗阻模型大鼠肾间质纤维化过程中血小板衍生生长因子D的表达

背景：血小板衍生生长因子在肾间质中通过诱导肾小管间质细胞增生、表型转化、炎性细胞浸润等导致肾小管间质纤维化。 目的：观察血小板衍生生长因子D在单侧输尿管梗阻模型大鼠肾脏组织中的表达水平及随时间的演变情况。 方法：将成年健康雄性SD大鼠60只随机分为模型组及假手术组,将模型组大鼠左侧输尿管结扎剪断建立单侧输尿管梗阻模型,假手术组大鼠不结扎剪断仅游离左侧输尿管。术后3,7,14,21,28 d,通过免疫组化检测血小板衍生生长因子D在肾脏组织中的表达分布情况,实时荧光定量RT-PCR方法检测血小板衍生生长因子D mRNA的表达水平及变化。 结果与结论：假手术组血小板衍生生长因子D仅少量表达于肾小球系膜细胞及血管平滑肌细胞,而在模型组,血小板衍生生长因子D同时表达于肾间质纤维化区域,随纤维化程度加重,表达增多。同时模型组血小板衍生生长因子D mRNA表达量较假手术组显著增多(P < 0.05),且表达随时间延长逐渐增多。提示血小板衍生生长因子D在单侧输尿管梗阻模型肾间质纤维化过程中发挥着促纤维化的重要意义。     

Expression of vascular endothelial growth factor and vascular endothelial growth factor receptor-2 (KDR/Flk-1) in ischemic skeletal muscle and its regeneration

Vascular endothelial growth factor (VEGF) is a hypoxia-inducible endothelial cell mitogen and survival factor. Its receptor VEGFR-2 (KDR/Flk-1) mediates these effects. We studied the expression of VEGF and VEGFR-2 in ischemic human and rabbit skeletal muscle by immunohistochemistry and in situ hybridization. Human samples were obtained from eight lower limb amputations because of acute or chronic critical ischemia. In chronically ischemic human skeletal muscle VEGF and VEGFR-2 expression was restricted to atrophic and regenerating skeletal myocytes, whereas in acutely ischemic limbs VEGF and VEGFR-2 were expressed diffusely in the affected muscle. Hypoxia-inducible factor-1alpha was associated with VEGF and VEGFR-2 expression both in acute and chronic ischemia but not in regeneration. Hindlimb ischemia was induced in 20 New Zealand White rabbits by excising the femoral artery. Magnetic resonance imaging and histological sections revealed extensive ischemic damage in the thigh and leg muscles of ischemic rabbit hindlimbs with VEGF expression similar to acute human lower limb ischemia. After 1 and 3 weeks of ischemia VEGF expression was restricted to regenerating myotubes and by 6 weeks regeneration and expression of VEGF was diminished. VEGFR-2 expression was co-localized with VEGF expression in regenerating myotubes. Macrophages and an increased number of capillaries were associated with areas of ischemic muscle expressing VEGF and VEGFR-2. In conclusion, two patterns of VEGF and VEGFR-2 expression in human and rabbit ischemic skeletal muscle are demonstrated. In acute skeletal muscle ischemia VEGF and VEGFR-2 are expressed diffusely in the affected muscle. In chronic skeletal muscle ischemia and in skeletal muscle recovering from ischemia VEGF and VEGFR-2 expression are restricted to atrophic and regenerating muscle cells suggesting the operation of an autocrine pathway that may promote survival and regeneration of myocytes.     

Involvement of neutrophil gelatinase-associated lipocalin and osteopontin in renal tubular regeneration and interstitial fibrosis after cisplatin-induced renal failure

The kidney has a capacity to recover from ischemic or toxic insults that result in cell death, and timely tissue repair of affected renal tubules may arrest progression of injury, leading to regression of injury and paving the way for recovery. To investigate the roles of neutrophil gelatinase-associated lipocalin (NGAL/lcn2) and osteopontin (OPN/spp1) during renal regeneration, the expression patterns of NGAL and OPN in the cisplatin-induced rat renal failure model were examined. NGAL expression was increased from day 1 after injection; it was seen mainly in the completely regenerating proximal tubules of the cortico-medullary junction on days 3–35; however, the expression was not seen in abnormally dilated or atrophied renal tubules surrounded by fibrotic lesions. On the other hand, OPN expression was increased from day 5 and the increased expression developed exclusively in the abnormal renal tubules. NGAL expression level well correlated with the proliferating activity in the regenerating renal epithelial cells, whereas OPN significantly correlated with the α-smooth muscle actin-positive myofibroblast appearance, expression of transforming growth factor (TGF)-β1, and the number of CD68-positive macrophages. Interestingly, rat renal epithelial cell line (NRK-52E) treated with TGF-β1 decreased NGAL expression, but increased OPN expression in a dose-dependent manner. Because increases of TGF-β1, myofibroblasts and macrophages contribute to progressive interstitial renal fibrosis, OPN may be involved in the pathogenesis of fibrosis; on the contrary, NGAL may play a role in tubular regeneration after injury. Expression analysis of NGAL and OPN would be useful to investigate the tubule damage in renal-toxicity.     

Chronic rejection of rat renal allograft. III. Ultrastructure of vascular and glomerular changes.

The main histological features for 'chronic' rejection of rat renal allograft are ongoing perivascular and interstitial inflammation, vascular intimal hyperplasia and glomerular sclerosis. In this communication we have investigated the ultrastructural features of renal parenchyma by comparing chronically rejecting rat renal allografts immunosuppressed initially with cyclosporine to similarly immunosuppressed syngeneic transplants. Electron microscopy confirmed that the primary arterial change was smooth muscle cell proliferation in media, thickening and focal destruction of the internal elastic lamina, and influx of smooth muscle cells and their proliferation in the intima, leading to a nearly complete occlusion of the lumen. In the glomeruli, the most prominent features were mesangial matrix increase, narrowing of capillary lumen, and basement membrane thickening with a double contour appearance in capillary walls. A thin membrane, similar to lamina densa, appeared beneath the endothelium in some segments. Mesangial interposition, formation of new basement membrane and mesangial widening due to increase of basement membrane-like material was nearly always present. As none of these vascular and glomerular changes were observed in similarly immunosuppressed syngeneic transplants, we consider these ultrastructural alterations characteristic for chronic rejection in a rat renal allograft.     

NADPH oxidases in the kidney

NADPH oxidases have a distinct cellular localization in the kidney. Reactive oxygen species (ROS) are produced in the kidney by fibroblasts, endothelial cells (EC), vascular smooth muscle cells (VSMC), mesangial cells (MCs), tubular cells, and podocyte cells. All components of the phagocytic NADPH oxidase, as well as the Nox-1 and -4, are expressed in the kidney, with a prominent expression in renal vessels, glomeruli, and podocytes, and cells of the thick ascending limb of the loop of Henle (TAL), macula densa, distal tubules, collecting ducts, and cortical interstitial fibroblasts. NADPH oxidase activity is upregulated by prolonged infusion of angiotensin II (Ang II) or a high salt diet. Since these are major factors underlying the development of hypertension, renal NADPH oxidase may have an important pathophysiological role. Indeed, recent studies with small interference RNAs (siRNAs) targeted to p22( phox ) implicate p22( phox ) in Ang II-induced activation of renal NADPH oxidase and the development of oxidative stress and hypertension, while studies with apocynin implicate activation of p47( phox ) in the development of nephropathy in a rat model of type 1 diabetes mellitus (DM). Experimental studies of the distribution, signaling, and function of NADPH oxidases in the kidney are described.     

Role of atrophic tubules in development of interstitial fibrosis in microembolism-induced renal failure in rat

We explored the origin and participation of atrophic tubules in the progression of interstitial fibrosis using a new microembolic rat model of chronic renal failure in which foci of atrophic tubules with cuff-like basement membrane thickening developed at 4 weeks. Atrophic tubules, immunoreactive for vimentin and platelet-derived growth factor, were surrounded by transformed interstitial cells expressing platelet-derived growth factor receptor beta and alpha-smooth muscle actin. Some tubules in the deep cortex and the outer stripe of the outer medulla had a mosaic appearance. Tall, intact proximal tubular cells with a brush border and positivity for Phaseolus vulgaris erythroagglutinin, adjoined typical atrophic tubule cells having no brush border and an immunostaining pattern characteristic for atrophic tubules. The transformed interstitial cells expressing alpha-smooth muscle actin were located near atrophic but not intact tubular epithelial cells. Type IV collagen accumulated between damaged tubular cells and transformed interstitial cells. Heat shock protein 47 showed immunoreactivity in damaged epithelial cells and in interstitial myofibroblasts. Staining with an anti-endothelial antibody suggested damage to peritubular capillaries near atrophic tubules. By disturbance of microcirculation following microsphere injection, proximal tubular cells expressed vimentin and platelet-derived growth factor; diffusion of the latter presumably stimulated transformation of interstitial cells to myofibroblasts. Injured tubular epithelial cells and interstitial myofibroblasts both were responsible for interstitial fibrosis.     

Induction of proinflammatory cytokines and nitric oxide by Trypanosoma cruzi in renal cells

Chagas disease is typically associated with cardiac involvement. During the acute phase of murine infection with Trypanosoma cruzi, severe acute myocarditis can develop. Prior to cardiac alteration, however, infected mice present with renal inflammatory infiltration causing acute kidney injury due to an ischemia/reperfusion lesion. Thus, the present study was undertaken in order to evaluate whether the parasites or some of their components would directly affect renal cells. As such, this study employed kidney cell lines (mesangial, epithelial, and proximal tubular) that mimic different regions of the renal system. Mesangial cells are more resistant to infection, showing reduced parasite internalization relative to epithelial and proximal tubular cells. Upon infection, mesangial cells produced more nitric oxide, tumor factor necrosis-α, and interferon-γ and showed decreased viability when compared to the other cell lines. These results indicate that the resistance of mesangial cells to infection may be related to the increased expression of nitric oxide and proinflammatory cytokines. Conversely, the high levels of nitric oxide produced by these cells caused impairment of cell integrity and viability. Higher nitric oxide concentrations promote cellular injury and can be involved in the genesis of ischemia/reperfusion lesions in acute kidney injury.